This invention relates to a process and diagnostic device for the determination of ammonia and of substrates which react with the formation of ammonia.
The determination of urea in body fluids is of great importance for the diagnosis and monitoring of diseases of the kidney. For some time now, a number of wet chemical processes have been available for this purpose which depend upon the following principles: In the case of a direct method, urea is reacted with diacetylmonoxime and the adsorption of the resulting compound is measured photometrically. Other more modern methods have, prior to the color reaction, an enzymatic step in which the urea is decomposed to ammonium carbonate by means of the enzyme urease. The ammonia is subsequently reacted, for example in the known Nessler color reaction or is converted according to berthelot's method, with phenol/hypochlorite, into a colored indophenol. The extinction of the colored reaction products formed by these reactions is finally a measure of the amount of urea initially present.
These processes are themselves of sufficient exactitude but, nevertheless, require the use of expensive photometers and trained personnel with sufficient experience in precise pipetting and in handling reagents, some of which are of limited stability and are also corrosive. Since these processes cannot be applied to whole blood, additional laboratory devices are necessary in order to obtain serum or plasma.
In emergency cases, for example in cases of uraemic coma, it is essential for the therapeutic measures which have to be taken that an accurate analytical result be available in the shortest possible time. Therefore, extensive efforts have also been made to develop methods for the determination of urea in body fluids which enable analysis results to be obtained in the shortest possible time which are of good diagnostic value.
Rapid tests in the form of test strips have recently achieved importance because of simplicity of handling and, in some cases, also the shorter reaction time, as well as the usability at the sick bed and in emergencies, and also use by untrained personnel or even by the patients themselves.
A rapid test for the determination of urea in body fluids, such as blood, serum and plasma, is described in German Pat. No. 1,240,306. In this case, urease, pH indicator, buffer and adjuvants are impregnated together onto a paper. Upon dropping a body fluid thereon, neutral-reacting urea contained therein is converted into ammonium carbonate, which has an alkaline reaction. The change in pH is indicated by a change in color of the pH indicator and the urea content of the sample can be estimated by comparison with comparative colors. In spite of certain advantages, such as ease of handling and a short reaction time, these test strips can only be used for rough estimations because of the difficulty of differentiating their reaction colors and because of the marked influence of the acid-base content of the body fluid.
The problem of dependence upon the acid-base content of body fluids is solved by strips described in German Pat. No. 1,245,619 in which an absorbent paper strip is impregnated side-by-side with three different solutions. in the case of these test strips, the urea-containing solution is first absorbed into a urease-containing test zone in which the urea reacts to give ammonium carbonate. The solution is then transferred by capillary forces to a neighboring alkaline test zone which liberates gaseous ammonia from the ammonium carbonate which passes over the gas space surrounding the test strips into a third test zone and colors a pH indicator present therein corresponding to the concentration of the urea. Transmission of the alkaline solution is prevented by means of an approximately 2 mm. wide hydrophobic zone between the second and third reaction zones. Since, in this manner, only gaseous ammonia passes over to the third reaction zone, other bases and acids present do not disturb the reaction. Furthermore, by means of buffering, the first test zone can be adjusted to an optimum pH value for urease so long as the degree of alkalinity of the second test zone is sufficient to liberate the ammonia quantitatively.
In spite of results which are of good diagnostic value, these test strips suffer from a number of disadvantages. The determination can only be carried out with serum or plasma but not with blood because the blood corpuscles disturb the chromatography. This necessitates centrifuging the blood, i.e., a large sample is needed and can only be obtained by a physician or appropriately well-trained personnel. In order to make the transmission of the ammonia through the gas space reproducible, the test strips must, in addition, be suspended in a special reaction chamber and precisely fixed relative to the sample during the reaction period. For the chromatography in the first and second reaction zones and especially for the diffusion of the ammonia through the gas space, a reaction time of 30 minutes is necessary, so that the test strips can scarcely be called rapid tests.
In German Pat. No. 2,249,647, there is described a similarly constructed test strip which possesses some constructional improvements. In this case, determinations can also be carried out on whole blood. However, in normal cases, these test strips also require a reaction time of 30 minutes and also require the use of a reaction chamber.
A further disadvantage of both of the above-mentioned test strips is the fact that the concentration of the urea takes place with a visual longitudinal measurement of the colorchanging zone of the indicator paper with an unclear boundary zone which is difficult to assess and cannot be carried out by the visual or precise measurement of the color depth with a remission photometer.
In German Pat. No. 2,626,367, there is described a device with several layers lying one on top of the other. For the detection of urea, these consist of a urease-containing layer, an alkaline buffer-containing layer and an indicator layer for detecting gaseous ammonia. In order to prevent the undesired diffusion of sample fluid from the urease layer to the indicator zone in addition to the gaseous ammonia, the urease layer and the indicator layer lying thereunder are separated by a hydrophobic, gas-permeable film or the indicator layer is made in the form of a hydrophobic film. The indicator layer, the intensity of the color of which differs according to the urea content of the sample, is measured through a carrier film and the concentration of the urea is thus determined.
In order to achieve reaction times of about 10 minutes, this test device must be incubated at 40.degree.-50.degree. C. because of the relatively high impermeability of the separating layer. Because of the cost of apparatus which this involves, it is not possible to use this test device as a rapid test.
Thus, all the previously known test strips for the determination of urea suffer from substantial disadvantages.
The present invention provides a device for the detection of urea in body fluids which: is simple to handle and does not require the additional use of a reaction chamber; enable a urea determination to be carried out not only in serum or plasma but also in small amounts of whole blood; at ambient temperature requires a reaction time of at most 10 minutes and preferably of about 5 minutes; gives semiquantitative results in the case of visual assessment; and in the case of remission photometric evaluation, enables a quantitative determination of urea to be carried out.